Promoting collateral formation in type 2 diabetes mellitus using ultra-small nanodots with autophagy activation and ROS scavenging

Background Impaired collateral formation is a major factor contributing to poor prognosis in type 2 diabetes mellitus (T2DM) patients with atherosclerotic cardiovascular disease. However, the current pharmacological treatments for improving collateral formation remain unsatisfactory. The induction of endothelial autophagy and the elimination of reactive oxygen species (ROS) represent potential therapeutic targets for enhancing endothelial angiogenesis and facilitating collateral formation. This study investigates the potential of molybdenum disulfide nanodots (MoS2 NDs) for enhancing collateral formation and improving prognosis. Results Our study shows that MoS2 NDs significantly enhance collateral formation in ischemic tissues of diabetic mice, improving effective blood resupply. Additionally, MoS2 NDs boost the proliferation, migration, and tube formation of endothelial cells under high glucose/hypoxia conditions in vitro. Mechanistically, the beneficial effects of MoS2 NDs on collateral formation not only depend on their known scavenging properties of ROS (H2O2, •O2-, and •OH) but also primarily involve a molecular pathway, cAMP/PKA-NR4A2, which promotes autophagy and contributes to mitigating damage in diabetic endothelial cells. Conclusions Overall, this study investigated the specific mechanism by which MoS2 NDs mediated autophagy activation and highlighted the synergy between autophagy activation and antioxidation, thus suggesting that an economic and biocompatible nano-agent with dual therapeutic functions is highly preferable for promoting collateral formation in a diabetic context, thus, highlighting their therapeutic potential. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1186/s12951-024-02357-z.


Background
Type 2 diabetes mellitus (T2DM) accelerates the progression of atherosclerotic cardiovascular diseases (ASCVDs).ASCVDs, including coronary artery disease and peripheral artery disease, lead to cardiac dysfunction and lower limb amputation due to occlusions of arteries [1,2].Studies suggest that when an artery is occluded, collaterals -the arteriolar networks connecting arteries in tissues such as the heart and skeletal muscle -serve as a conduit, which can partially ensure blood supply, prevent or alleviate tissue ischemia, and reduce complications [3][4][5].However, collaterals in patients with T2DM are also weaker than those in non-diabetic patients due to more severe and diffused artery atherosclerosis and impaired endothelial cell function [6,7].
Impaired collateral formation leads to poor prognosis of T2DM patients with ASCVDs [8].Attempts to improve collateral formation in diabetes have failed to achieve the expected results [9,10].Pro-angiogenic factors, including vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF), are recognized as highly effective substances in promoting collateral formation [9,11].However, under diabetic conditions, there is an impairment of angiogenic signaling pathways, accompanied by the overactivation of the ubiquitin-proteasome pathway and the downregulation of their respective receptors [12,13].These changes result in the suboptimal efficacy of pro-angiogenic factorbased therapies.Furthermore, cell-based therapies [14] aiming to promote collateral formation also encounter obstacles.Factors such as hyperglycemia, ischemia, and inflammation within the diabetic environment can negatively impact the survival and functionality of transplanted cells, which leads to significant impairments in the effectiveness of stem cell-mediated repair [15].Therefore, the exploration and development of new drugs and approaches to address the negative impact of the diabetic environment and endothelial dysfunction to enhance collateral formation are urgently needed.
High glucose and advanced glycation end products (AGEs) accumulation lead to an overload of reactive oxygen species (ROS) [16], and inhibition of cellular autophagy [17], representing the two primary pathological factors underlying endothelial cell dysfunction in diabetes.ROS overload leads to cellular inflammatory response [18], DNA damage [19], and lipid peroxidation in the cell membrane [20], ultimately triggering endothelial cell apoptosis.Autophagy inhibition leads to the failure to clear damaged substances [21], intensifying intracellular oxidative stress and inflammation [22,23], impacting cellular energy metabolism [24], and disrupting the normal physiological functions of endothelial cells, including their survival, proliferation, migration, and sprouting capabilities [25].Therefore, reducing ROS levels and activating autophagy both play roles in maintaining the homeostasis of endothelial cells, thereby protecting endothelial cell function and improving collateral formation [26,27].
Inorganic nanoparticles have been developed and utilized in therapy for various diseases owing to their outstanding advantages, including high stability, long blood half-life, and multiple enzyme-like activities compared with traditional medication [28][29][30].As a two-dimensional transition metal dichalcogenide, molybdenum disulfide (MoS 2 ) nanosheets have found widespread applications in anti-inflammatory and antibacterial therapies [31].To enhance the biocompatibility of MoS 2 materials, sub-10-nanometer ultra-small MoS 2 nanodots (MoS 2 NDs) have been developed [32,33].These nanodots not only retain the distinctive ROS scavenging properties of MoS 2 [34] but also demonstrate higher clearance rates and less retention in vivo [35,36].It is noteworthy that recent studies have revealed the significant enhancement of cellular autophagy activity by MoS 2 nanomaterials [37,38].Considering the crucial roles of ROS overload and autophagy inhibition in diabetic vascular diseases, we speculate that MoS 2 nanodots with dual functions in ROS scavenging and autophagy activation may serve as effective means to intervene in diabetic collateral formation [39,40].Therefore, we prepared MoS 2 NDs using a simple solvothermal method and assessed their characteristics, such as body clearance rate, biocompatibility, and catalytic properties.To comprehensively assess the effect of MoS 2 NDs on collateral formation in diabetes, we established diabetic mouse hindlimb ischemia (HLI) models and high glucose/hypoxia (HG/Hypo) endothelial cell models treated with MoS 2 NDs.Various indicators, including angiogenic capacity after ischemia, endothelial cell function, and autophagic activity, were examined.Additionally, by using omics analysis and molecular biology techniques, we further revealed a novel molecular mechanism by which MoS 2 NDs activate endothelial autophagy, involving the upregulation of the cAMP/PKA-NR4A2 pathway.Overall, this study is expected to provide potential therapeutic approaches for treating impaired collateral formation in diabetes (Fig. 1).

Animal feeding
C57BL/6 mice (males, 7-8 weeks) were obtained from the Shanghai JASJ Laboratory Animal Co.All animal experiments were performed in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85 − 23, revised 1996) and were approved by the Animal Resources Committee of Shanghai Jiao Tong University.All mice were housed in a pathogen-free setting at the Animal Experimentation Center of Ruijin Hospital, Shanghai Jiao Tong University School of Medicine.The animals were given free access to food and water during the 12 h light/dark cycle.

MoS 2 NDs preparation
MoS 2 NDs were synthesized according to a previously described method [35].Briefly, 150 mg of polyvinyl pyrrolidone (PVP) (MW: 10,000, Cas# 9003-39-8, Sigma-Aldrich, USA) and 25 mg of ammonium tetrathiomolybdate [(NH 4 ) 2 MoS 4 ] (Cas# 15060-55-6, Sigma-Aldrich) were mixed in 80 mL of methanol.Next, 0.80 mL of hydrazine hydrate (Cas# 10217-52-4, Sigma-Aldrich) was added dropwise.The mixture was ultrasonicated for 1 h, then placed in a stainless-steel autoclave lined with Teflon, and was allowed to react at 120 °C for 3 h.Subsequently, the dispersion was filtered, and methanol was removed using a rotary evaporator.The final dialyzable dispersion was obtained by dialyzing the result against deionized water six times for 72 h.The morphology of MoS 2 NDs was assessed by Transmission electron microscopy (TEM) (JEM-2100 F, Japan Electronics Co., Ltd.).D8 ADVANCE X-ray Diffraction Instrument (Bruker, USA) was used to measure the X-ray Diffraction (XRD) pattern of MoS 2 NDs.An ESCAlab250 electron spectrometer (Thermo Fisher Scientific, USA) was used to obtain X-ray photoelectron spectroscopy (XPS).The hydrodynamic sizes of the nanoparticles were characterized using a Nano ZS 90 Malvern Zetasizer Nano Series (Malvern, UK).An Evolution 350 (Thermo Fisher Scientific, USA) was used to obtain the UV-vis absorption spectra of the MoS 2 NDs.For fluorescein isothiocyanate (FITC) labeling, MoS 2 NDs aqueous solution (10 mL) was mixed with an ethanolic solution of FITC-PEG-SH (0.1 mg/mL).The mixture was stirred for 12 h in the dark, followed by dialysis against deionized water for 3 days to obtain the FITC-labeled MoS 2 NDs.

Hemolysis assay
Blood was mixed with PBS (10 mM) and then centrifuged at 3000 rpm for 5 min.This process is repeated 3-4 times to get erythrocytes.The precipitated erythrocytes were re-dispersed in PBS and then added to of pure water (positive control), PBS (negative control), and different concentrations of MoS 2 NDs.After that, the mixtures were cultured at 37 °C for 8 h.Subsequently, the absorbance of supernatants at 540 nm was measured by UV − vis spectra.The hemolysis rate (HR%) was calculated according to the following formula: HR% = (A MoS2 -A PBS )/(A water -A PBS ) × 100%.Where A MoS2 , A water , and A PBS are the absorbance of the sample, the positive control, and the negative control, respectively.

In vivo biodistribution and metabolism study
Tail vein injections of the MoS 2 NDs were performed 0.5, 2, 6, 12, and 24 h after injections.The hearts, livers, spleens, lungs, and kidneys were removed 0.5, 2, 6, 12, and 24 h after injection.A lysis buffer of 0.5 mL was applied after the trituration of all collected tissues, and the Mo level was determined using ICP-OES.To evaluate the in vivo metabolism of the MoS 2 NDs, mouse urine and feces were collected at different times and analyzed using ICP-OES.The distribution and metabolism in different organs, feces, and urine were expressed as percentages of the tissue-injected dose per gram (%ID/g).To examine the shape of MoS 2 NDs in urine and feces, we collected them from 24 to 48 h after the injection of MoS 2 NDs.urine samples from mice were centrifuged at 12,000 rpm for 10 min.The pellet was dissolved in water, and a droplet was placed on a copper grid to observe the morphology of MoS 2 NDs by TEM.Fecal samples were collected and made into a suspension.The suspension was centrifuged first at 1000 rpm for 10 min to obtain a supernatant.The supernatant was further centrifuged at 12,000 rpm for 10 min.The resulting pellet was dissolved in water.A droplet of the suspension was placed on a copper grid for visualization.

Femoral artery ligation
HFD/STZ-induced diabetic mice were anesthetized with continuous inhalation of 2.5% isoflurane gas.The left superficial femoral artery was ligated proximal to the deep femoral artery and was ligated at the branch to the tibial artery, clipping the ligature short and leaving the nerve and vein intact.Subsequently, the blood flow during functional recovery was detected by the laser Doppler imaging instruments (MoorLDI2, Moor, UK) on days 0, 3, 7, 14, and 21.The recovery rate was measured as the blood flow on the affected and unaffected sides [41].The limb function score was used to assess ischemic injury and mobility impairment semi-quantitatively.It was calculated as follows: ischemic damage: contralateral hindlimb = 0; mild discoloration = 1; moderate discoloration = 2; severe discoloration or partial tissue loss = 3; and any amputation = 4 [42].Mice were injected with MoS 2 NDs (2 mg/kg) and PBS through the tail vein every other day from 3 days preoperatively to 21 days postoperatively.Cy5.5-MoS 2 NDs were synthesized to assess the biodistribution of MoS 2 NDs in ischemic muscles in the hindlimb ischemia model 3 days preoperatively by the in vivo fluorescence imaging system (CleVue Basic System, Vieworks, Korea).The biodistribution of MoS 2 NDs on the ischemic site and the non-ischemic site was measured by ICP-OES.

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay
The TUNEL assay was conducted based on the manufacturer's protocol for the Click-iT TUNEL assay (Cat# C10245, Invitrogen), and the cells were counterstained with DAPI for 10 min before washing.Images were captured using a fluorescence microscope, and the positive areas were counted using the ImageJ software.

Dihydroethidium (DHE) staining
DHE (Cat# S0063, Beyotime) staining was used to evaluate ROS generation in the calf muscles.A cutting temperature compound was used to embed calf muscle samples in Tissue-Tek.Then, skeletal muscle cryosections (10 mm) were incubated for 30 min with DHE.Finally, images were obtained using fluorescence microscopy, and DHE-positive areas were counted using ImageJ software.

Malondialdehyde (MDA) and SOD activity assay
MDA and SOD activity measurements were conducted as directed by the manufacturer for the MDA assay kit (Cat# BC0025, Solarbio, China) and SOD assay kit to detect the MDA and SOD levels of the serum of diabetic mice.

Cell stimulation
We created HG/Hypo conditions by adding 33.3 mM glucose to the endothelial cell medium with 1% FBS, and the Anaeropack system (Cat# D-07, Mitsubishi Gas Chemical Co, Japan) was used to create a hypoxia condition.Next, HUVECs in a complete endothelial cell medium with 5.5 mM glucose without the Anaeropack system were cultured as the control group (normal glucose, NG).After HUVECs were cultured for 2 days under NG and HG/Hypo conditions, MoS 2 NDs (50 µg/mL) or PBS were added to the culture medium for 12 h for subsequent experiments.HUVECs were incubated with Baf A1 (1 nM) (Cat# HY-100558, MedChemExpress, USA), 3-MA (Cat#HY-19132, MedChemExpress, USA) and SQ22536 (250 µM) (Cat# S8283, Selleck, USA) to inhibit autophagy and cAMP level, respectively.

Wound healing assay
HUVECs were pretreated until they reached 100% confluence, and an artificial gap was generated using a 200 µL micropipette tip.Images were acquired using inverted microscopy (IX71, Olympus) immediately after the scratches were generated and 24 h later.Finally, the cell migration area was quantified by the closure of the scratch area.

Transwell assay
HUVECs were seeded onto each Transwell inserts (Cat# 3422, Corning, USA).A culture medium of 500 L was added to the lower chamber.We removed the medium after 12 h and fixed them for 30 min.Subsequently, each well was stained for 20 min with 0.2% crystal violet.After removing the stain, the cells were photographed by the inverted microscope.

Tube formation
Matrigel (Cat# 356,234, Corning) was added before solidifying at 37 °C for 20 min, and the cell solution (100 µL) with 2 × 10 4 cell numbers was added to the gel and incubated for 4 h.Next, the tubes were photographed using an inverted microscope, and the vessel length and number were counted using the ImageJ software.

Immunofluorescence staining
We seeded HUVECs on 24-well plates, fixed them with 4% PFA, and incubated them with anti-p16 antibodies

Annexin V-FITC/propidium iodide (PI) assay
An annexin V-FITC/PI assay kit (Cat# 521942, Solarbio) was used for this experiment.Following pretreatment with Annexin V-FITC in the dark for 20 min, HUVECs are resuspended in the binding buffer and analyzed by flow cytometry.

5-Ethynyl-2-deoxyuridine (EdU) assay
HUVECs were incubated with 10 µM EdU (Cat# C10310, Riobobio, China) for 6 h, with a fixation for 30 min and a permeabilization for 20 min (0.5% Triton X-100) before the Click-iT reaction cocktail (Cat# C10310-1, Riobobio) for 45 min at room temperature.A DAPI counterstain was applied to the cells, which were photographed using fluorescence microscopy, and EdU + cells were counted using the ImageJ software.

Cell transfection
Autophagy activation was measured using lenti-mRFP-GFP-LC3 (Cat# GM-1314l204H, Genomeditech, China).Autolysosomes are indicated in the presence of co-localized GFP and RFP fluorescence, and autolysosomes are indicated in the absence of GFP.Transfection efficiency was determined using fluorescence microscopy and laser scanning confocal microscopy (LSCM) (Sted 8; Leica, Germany).
NR4A2 small interfering RNA was obtained from Genomeditech (Shanghai, China).The sequences are as follows: 5 Si-Ctrl or si-NR4A2 was transfected with 50 nM using lipofectamine3000 (Cat# L3000075; Invitrogen), as per the manufacturer's instructions.The effects of NR4A2 knockdown were also assessed by western blotting.

Cell cycle assay
After the pretreatment, cells were collected with a fixation in 70% ethanol at 4 °C overnight, followed by staining with PI (Cat# 1052, Beyotime), staining solution in the dark for 30 min, and performing flow cytometry measurement.We determined the percentage of cells in the G1, S, and G2 phases using FlowJo software (BD Biosciences, USA).

Quantitative reverse transcription polymerase chain reaction (RT-qPCR)
RT-qPCR was performed to determine the expression of NR4A2, FOXM1, SOX6, and ZMIZ1 Briefly, and total RNA was isolated from HUVECs using TRIzol Reagent (Cat# 15596026, Invitrogen).The extracted RNA was reverse-transcribed (Cat# RR036A, Takara, Japan).RT-qPCR was conducted using a TB Green RT-qPCR Kit (Cat# RR820A, Takara) to determine the expression of the target genes.All primers used are listed in Table S3.

Transcriptome analysis
Total RNA was extracted from HUVECs after pretreatment and harvesting.A Qubit Fluorometer (Qubit Flex, Thermo Fisher Scientific) and Tapestation (4200 Tapastation, Agilent) were used to measure the RNA quality and quantity.Sequencing libraries were prepared using the SMARTer Universal Low Input RNA Kit (Takara).
Mammalian-specific R-probes were used to cleave ribosomal cDNA using ZapR.PCR primers matching the Illumina adapters were used to enrich the remaining fragments.The libraries were normalized to 10 nM Tris-HCl (10 mM, pH 8.5) containing 0.1% Tween 20.To map and trim the FASTQ format, A TrimGalore sequence was generated and a FastQC-based quality assessment was conducted on the sequences.A Hisat2 aligner was used for alignment with the Ensembl reference genome, GRCm38.Gene expression was calculated using the R package 'feature counts' (R Foundation, New Zealand).
Before the analysis, we used a cutoff of 1 to define the minimum level of expression for each gene, and a negative binomial model was implemented in the Bioconductor package DESeq to calculate differential gene expressions.Significantly differentially expressed genes were defined as having P < 0.05, and pathway analysis was performed using the Cluster Profiler R package.

Chromatin immunoprecipitation sequencing (ChIP-seq) analysis
ChIP-seq data for NR4A2 were downloaded from the Gene Expression Omnibus database (GSE186197).The R package "ChIPseeker" was used to depict the positions of NR4A2-binding peaks on genome-wide scales.The sequenced reads were mapped to the human genome (hg38) using Integrative Genomics Viewer.De novo DNA motif analysis and construction from the ChIP-seq data were performed using MEChIP.

Statistical analysis
The mean and standard deviations of all data were calculated using GraphPad Prism version 9.0 (GraphPad Software, USA).One-way analysis of variance (ANOVA), two-way ANOVA, and t-test were used for comparisons, and signs of significance were defined as P < 0.05.

MoS 2 NDs preparation and characterization
MoS 2 NDs were developed using a one-step solvothermal process, wherein ammonium tetrathiomolybdate [(NH 4 ) 2 MoS 4 ] was dissolved in a reducing environment (hydrazine hydrate) at 120 °C for 3 h (Fig. 1) [35].To control the particle size and stabilize the MoS 2 ND, PVP was introduced as a dispersion stabilizer during the preparation process.TEM images showed that the MoS 2 NDs exhibited a uniform sphere shape with a diameter of approximately 2.2 nm (Fig. 2A).The high-resolution TEM (HRTEM) image (inset) of MoS 2 NDs illustrated the hexagonal lattice structure with a lattice space of 2.7 Å, which was assigned to the (100) planes of the typical hexagonal structure of MoS 2 [43].The XRD pattern also demonstrated the good crystallinity of MoS 2 NDs (Figure S1).Additionally, they dispersed well in water with a hydrodynamic size of 13.5 ± 2.3 nm (Fig. 2B) and a zeta potential of -16.5 mV.The size difference of the MoS 2 NDs determined by dynamic light scattering (DLS) and TEM mainly resulted from the absorption of water molecules since DLS performed wet samples [44].Notably, only when the hydrodynamic diameter of the nanoparticles is smaller than the glomerular filtration threshold (20 nm) of the kidney can nanomaterials be excreted through the renal clearance pathway, which exhibits exceptional biosecurity [45][46][47].MoS 2 NDs have also been demonstrated to have such potential, as reported by earlier investigations [35,36], which was consistent with our results.The UV-vis absorption spectra of the MoS 2 NDs are shown in Fig. 2C.Furthermore, the compositions of MoS 2 NDs were measured by XPS.The characteristic binding peaked at 228.6 eV, 230.0 eV, and 231.9 eV, which corresponded to the 3d 3/2 and 3d 5/2 peaks of Mo 4+ , respectively, and characteristic peaks located at 232.0 and 235.1 eV could be ascribed to the Mo 6+ .Moreover, the 161.7 eV and 163.4 eV peaks could be assigned to the S 2p 3/2 and 2p 1/2 (S 2-), respectively (Fig. 2D-F).Also, the strong 3d 3/2 and 3d 5/2 peaks of Mo (230.0,232.0, 233.1, and 235.1 eV) and the peak at 530.37 eV of O indicated the formation of the Mo-O bond (Figure S2, Table S1).These data demonstrate that Mo in the MoS 2 NDs had reduced (IV) and oxidized (VI) states, enabling it to perform redox interactions, which is consistent with previous studies [35,48].These data confirmed the successful MoS 2 NDs synthesis.Furthermore, MoS 2 NDs were dispersed in three media: deionized water (DI water), phosphate-buffered saline (PBS), and Dulbecco's modified Eagle's medium (DMEM), and stabilized after storing for at least a week, indicating their stability under physiological conditions (Figure S3).
As shown in Fig. 2G, bio-TEM revealed a large number of nanodots in the endothelial cells without significant damage, degradation, or deformation under normal glucose (NG) conditions.Moreover, by ICP-OES, the level of Mo dissolution was measured in endothelial cells.After 24 h incubation, the Mo level increased from 0.37 ± 0.03 pg/cell to 3.15 ± 0.12 pg/cell (Fig. 2H).This indicated that MoS 2 NDs could enter into endothelial cells and exhibited good stability within endothelial cells.Furthermore, to evaluate the in vitro uptake efficiency, FITC-labeled MoS 2 NDs were prepared by mixing MoS 2 NDs with FITC-PEG-SH under sonication and incubating at room temperature.Fourier-transform infrared spectroscopy (FT-IR) spectra confirmed the successful preparation of FITC-labeled MoS 2 NDs (Figure S4).Next, the FITClabeled MoS 2 NDs were incubated with HUVECs for 1, 2, 4, 6, 12, and 24 h.Fluorescence microscopy revealed that the FITC signal intensity increased over time and remained constant after 12 h (Fig. 2I).Moreover, the fluorescence intensity measured using flow cytometry was consistent with fluorescence microscopy (Fig. 2J), which indicated that the uptake of MoS 2 NDs by endothelial cells depended on the incubation duration.
Subsequently, in vivo biodistribution of MoS 2 NDs was measured in healthy mice.The liver and spleen exhibited the highest dose distributions, whereas the heart and lungs exhibited the lowest distribution (Figure S5A).Moreover, MoS 2 NDs rapidly accumulated in the kidney within 6 h and were subsequently excreted through the urine within 24 h, thereby indicating the effective renal excretion of the MoS 2 NDs.Next, the in vivo metabolism of MoS 2 NDs was examined by evaluating the Mo content in feces and urine at days post-administration.It was found that the feces and urine showed the highest Mo levels at days post-administration, which decreased over time (Figure S5B).We also characterized the morphology of MoS 2 NDs in feces and urine by TEM, confirming that MoS 2 NDs existed in feces and urine as their original form, further demonstrating the excellent metabolic capacity of MoS 2 NDs (Figure S6A-B).The biosafety of the MoS 2 NDs was confirmed using the CCK-8 assay on HUVECs (Figure S7), which showed no cytotoxicity from 25 to 200 µg/mL.No significant differences in the indexes of VCAM-1 and ICAM-1 in endothelial cells, as well as IFN-γ, IL-1β, IL-6, and TNF-α in healthy mice serum between the MoS 2 NDs and PBS-treated was noted, indicating MoS 2 NDs did not cause inflammatory responses both in vitro and in vivo (Figure S8-S9).Major organ histopathology showed no fibrosis, necrosis, or no hemorrhage in the lungs, livers, spleens, kidneys, or hearts 7 days after treatment with MoS 2 NDs (Figure S10).Furthermore, routine blood parameters (Figure S11A-D) and liver and kidney function (Figure S11E-F) were not significantly different between mice injected with PBS and MoS 2 NDs.The hemolysis rate of MoS 2 NDs at different concentrations was determined by using red blood cells (Figure S12).No obvious red color was observed even at the high concentration of 100 µg/mL-500 µg/mL (hemolysis rate < 5%), indicating the good blood compatibility of MoS 2 NDs.These results demonstrate the biosafety of MoS 2 NDs in vivo.These results also confirmed that the particle size and hydrodynamic diameter of MoS 2 NDs were sufficient for their metabolism through the kidney while exhibiting exceptional biosafety both in vivo and in vitro.
Subsequent experiments in this study focused on applying MoS 2 NDs in diabetic contexts, which led to oxidative stress [49] and a decrease in the pH level in the cytoplasm [50].Therefore, we checked the stability of MoS 2 NDs in acidic and oxidizing environments (pH 5.0, 200 mM H 2 O 2 ).There were no significant changes in the diameter distribution and absorption spectra after storage for 7 days, while a measure of biodegradation after 7 days of co-incubation (Figure S13).Additionally, the TEM image indicated that the structure and morphology of MoS 2 NDs remained intact integrally but showed slight structural changes on day 7 (Figure S14), thereby demonstrating the acceptable stability of the designed MoS 2 NDs in acidic and oxidizing environments.Moreover, HG/Hypo conditions did not affect the uptake behavior of MoS 2 NDs by endothelial cells (Fig. 2H), and bio-TEM also showed intact MoS 2 NDs within endothelial cells under HG/Hypo conditions (Fig. 2G).These results illustrated the stability of MoS 2 NDs and laid the foundation for their application in diabetes settings.

The antioxidative properties of the MoS 2 NDs
Reportedly, MoS 2 -based nanomaterials possess ROSscavenging capacity owing to their intrinsic enzyme-like activities (e.g., SOD, CAT, and peroxidase) [34,36].The possible mechanisms of the enzyme-mimicking properties involve switching the Mo (IV)/Mo (VI) redox couple on the surface of MoS 2 [48], where the Mo (VI) sites are responsible for the H 2 O 2 oxidation through CAT mimetics and Mo (IV) sites are used to scavenge •O 2 -and •OH (Fig. 3A).The •OH free radicals were determined with DMPO spin traps monitoring by EMX1598 ESR spectra.A distinct signal for the DMPO/•OH adducts was induced by the Fe 2+ /H 2 O 2 system (Fig. 3B).MoS 2 NDs effectively scavenged •OH, as indicated by the reduced signal intensity of DMPO/•OH, demonstrating the •OH scavenging capacity of the MoS 2 NDs (Fig. 3C).Additionally, the clearance ratio of •OH was dose-dependent, as determined by the •OH-specific probe orthophenanthroline (Fig. 3D).We also monitored the superoxide anion-decomposition ability of the MoS 2 NDs using the SOD assay (Fig. 3E).The scavenging rate increased from 16.8 to 76.7% at different concentrations, confirming that MoS 2 NDs exhibited SOD-like activity to quench •O 2 -.In addition, the CAT-like capacity of MoS 2 NDs was determined by the H 2 O 2 -specific probe xylenol orange.As shown in Fig. 3F, there was a significant increase in the clearance ratio of H 2 O 2 , sharply increasing from 35.9 to 95.2% with increasing concentrations of MoS 2 NDs.Meanwhile, visible bubbles (O 2 ) were produced in the presence of MoS 2 NDs and H 2 O 2 , which further indicated the CAT-like activity of MoS 2 NDs (Figure S15).Also, the XPS spectra of MoS 2 NDs were measured after reacting with H 2 O 2 to reflect the valence state changes (Figure S16).After the treatment with H 2 O 2 (50 mM), the peaks at 228.5 eV assigned to Mo 3d5/2 of Mo 5+ were shifted to higher intensities, and the Mo 3d5/2 and Mo 3d3/2 peaks at 231.8 and 234.8 eV of Mo 6+ were shifted to lower intensities, suggests the valence change of molybdenum element after the addition of H 2 O 2 .Overall, MoS 2 NDs exhibited robust CAT, SOD like activities, and •OH scavenging ability; they were used as an antioxidant for subsequent evaluation.Excessive ROS are generated  NDs = 4.35 ± 0.12 × 10 4 , P < 0.0001) (Fig. 3G-H), thereby confirming MoS 2 NDs as an effective intracellular ROS scavenger in diabetic context.
Overall, the conducted experiments have highlighted the outstanding biosafety, robust stability, remarkable efficiency in cellular uptake, and significant ROS scavenging ability of MoS 2 NDs.This not only distinguishes them from other forms of MoS 2 but also underscores their advantages compared with all other nanomaterials (such as silver nanoparticles, zinc oxide nanoparticles, and nanoceria) utilized in the treatment of diabetesrelated complications.

MoS 2 NDs improved collateral formation in diabetic mice
In the hindlimb ischemia model, the femoral arteries of mice are ligated.This process causes an increase in angiogenetic factors in the ischemic muscle [53].As a result, endothelial cells may be activated and mobilized, forming new leaky blood vessels, leading to the accumulation of nanoparticles caused by the enhanced permeability and retention (EPR) effect [54].We also further demonstrated the passive targeting of MoS 2 NDs owing to the EPR effect at ischemic muscles by in vivo fluorescence imaging.Cy5.5-MoS 2 NDs were injected through the tail vein, and the fluorescence signal increased with time.At 3-6 h after injection, the signal reached the peak, which decreased later owing to in vivo metabolism (Figure S17A).The fluorescence intensity was stronger at the ischemic site than at the non-ischemic site (Figure S17A).In addition, the CleVue Basic system was used to perform a semi-quantitative analysis of the fluorescence intensity of Cy5.5 at the ischemic site, which further validated the aforementioned findings (Figure S17B).Moreover, in vivo biodistribution of MoS 2 NDs in the ischemic site and the non-ischemic site was measured to further confirm the EPR effect at ischemic muscles (Figure S18).Therefore, MoS 2 NDs were shown to be targeted to the ischemic site, thus laying the groundwork for subsequent therapeutic studies in vivo.
Subsequently, to evaluate the effect of MoS 2 NDs on the restoration of blood flow and neovessels formation after HLI in HFD/STZ-induced type 2 diabetic mice, we treated the mice with PBS or MoS 2 NDs every other day from 3 days preoperatively to 21 days postoperatively (Fig. 4A).Although no changes were observed in blood glucose change (Table S2), the MoS The area of fibrosis was also significantly reduced, as indicated by Masson staining (Figure S20).Furthermore, the ischemia score at the aforementioned time points also revealed that MoS 2 NDs improved the blood supply (Figure S21).Next, the ROS scavenging ability of MoS 2 NDs was also confirmed in vivo, as indicated by the reduced DHE intensity in the muscle sections in the MoS 2 NDs group (Fig. 4G), as well as the decreased MDA level and increased SOD level in the serum of diabetic mice (Figure S22).The data showed that MoS 2 NDs had a positive impact on reducing oxidative stress in diabetic settings.Additionally, the number of CD31 + and α-SMA + vessels was significantly higher in the MoS 2 NDs group (Fig. 4H), indicating increased angiogenesis post-HLI in the MoS 2 NDs group.These findings provided strong evidence of MoS 2 NDs improving blood flow restoration and neovessel formation at the ischemic site after HLI in diabetic mice.

MoS 2 NDs improved endothelial cell angiogenic dysfunction induced by high glucose and hypoxia
Endothelial cells are determinants for the maintenance of vascular integrity and blood flow.During collateral formation, endothelial cells recruit pericytes, parietal cells, smooth muscle cells, and factors to form blood vessels [55,56].Therefore, restoring endothelial function and promoting neovascularization are effective strategies for treating diabetic vascular diseases and have become the focus of current research [57,58].Therefore, when MoS 2 NDs have gained therapeutic benefit in vivo, we focused our research on endothelial cells to explore the specific mechanism of how MoS 2 NDs alleviate endothelial dysfunction and thus promote angiogenesis.RNA sequencing analysis was conducted under HG/Hypo conditions in endothelial cells to identify the potential mechanisms of angiogenesis regulated by MoS 2 NDs.A total of 3311 genes were differentially expressed, 1684 of which were upregulated and 1627 were downregulated (Fig. 5A).These genes were enriched for multiple angiogenesis pathways such as PI3K-AKT [59], MAPK signaling pathways [60], and endothelial proliferation by Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis (Fig. 5B, Figure S23-S26).
To confirm the pro-angiogenic effects of MoS 2 NDs in vitro, we conducted tube formation, wound healing, and transwell assays.Tube formation showed that, under NG conditions, the total vessel length and vessel area per field between the PBS and MoS 2 NDs remained unchanged.In contrast, under HG/Hypo conditions, the total vessel length and vessel area significantly increased with the administration of MoS 2 NDs (Fig. 5C-E).Additionally, transwell and wound healing assays revealed that the migration area increased significantly with MoS 2 NDs administration under HG/Hypo conditions (Fig. 5F-I).Moreover, the intracellular nitric oxide (NO), a proangiogenic factor, was detected by DAF-FM-DA and showed increased amounts of NO upon treatment with MoS 2 NDs under HG/Hypo conditions (Figure S27).All the above data demonstrated the pro-angiogenic effects of MoS 2 NDs under HG/Hypo conditions in vitro.

MoS 2 NDs inhibited cell apoptosis induced by HG/Hypo and promoted endothelial cell proliferation
Endothelial proliferation is important for endothelial angiogenesis.Impaired angiogenesis can be attributed to decreased cell proliferation levels and increased apoptosis levels of endothelial cells under HG/Hypo conditions [61,62].To determine whether MoS 2 NDs could promote endothelial proliferation and inhibit endothelial apoptosis induced by HG/Hypo conditions, Gene Set Enrichment  Analysis (GSEA) was performed.As shown in Fig. 6A and  B, the GSEA and heatmaps indicated positive enrichment associated with proliferation and negative enrichment associated with apoptosis.Next, a set of experiments was conducted to confirm the effect of MoS 2 NDs on the proliferation and apoptosis of endothelial cells.We conducted a cell cycle assay and found that the treatment of MoS 2 NDs under HG/Hypo conditions affected the ratio of each cell cycle phase (G0G1, G2M, and S phases).The result showed that fewer endothelial cells were in the G0-G1 phase (HG/Hypo + PBS = 81.25 ± 4.45% vs. HG/ Hypo + MoS 2 NDs = 67.11± 4.87%, P = 0.006) and more in the S phase (HG/Hypo + PBS = 12.79 ± 3.25% vs. HG/ Hypo + MoS 2 NDs = 26.04 ± 3.82%, P = 0.042) in the MoS 2 NDs group under HG/Hypo conditions (Fig. 6C-D).Moreover, The CCK-8 assay revealed that the MoS 2 NDs (50 µg/mL) significantly promoted endothelial cell proliferation under HG/Hypo conditions (Figure S7), which is consistent with the result of EdU staining (Fig. 6E-F).These results indicated the potential of MoS 2 NDs to promote endothelial proliferation.Meanwhile, Annexin V/ PI flow cytometry revealed that MoS 2 NDs reduced the number of apoptotic endothelial cells under HG/Hypo conditions (Fig. 6G-H).The negative cell cycle regulators p16 and p21 were also evaluated by western blotting and Immunofluorescence staining.They were found significantly decreased in MoS 2 NDs under HG/Hypo conditions (p16: HG/Hypo + PBS vs. HG/Hypo + MoS 2 NDs = 1.50 ± 0.37, P = 0.046; p21: HG/Hypo + PBS vs. HG/Hypo + MoS 2 NDs = 2.66 ± 0.11, P < 0.001) (Fig. 6I-J, Figure S28).Additionally, the protein expression levels of apoptosis-related factors, Bcl-2, Bax, and cleaved   6K-L).To confirm the apoptotic effect, the JC-1 staining assay was also performed.The early stages of apoptosis are characterized by a decline in mitochondrial membrane potential, which was detected by the transient release of JC-1 from the aggregates to the monomers.The HG/Hypo conditions induced the release of JC-1 monomers, but MoS 2 NDs aggregated it again (Fig. 6M).All these results comprehensively confirmed that MoS 2 NDs could reduce the proportion of apoptotic endothelial cells under HG/Hypo conditions.Muscle sections also showed increased Ki67 + signaling and reduced signaling of TUNEL in the MoS 2 NDs group, confirming that MoS 2 NDs were involved in cell proliferation and apoptosis in vivo(Figure S29-S30).

MoS 2 NDs activated endothelial autophagy through the cAMP/PKA-NR4A2 signaling pathway
To explore the mechanisms by which MoS 2 NDs promoted autophagy under HG/Hypo conditions, we identified four autophagy-related TFs (ZMIZ1, SOX6, NR4A2, FOXM1) [70][71][72][73][74] that were upregulated among the differential genes according to the RNA-sequencing (Fig. 8A).RT-qPCR results showed that MoS 2 NDs upregulated the mRNA levels of all four genes in a dose-and time-dependent manners (Fig. 8B, Figure S36-S37), with NR4A2 exhibiting the highest increases.Immunofluorescence staining revealed that NR4A2 was highly expressed in the nuclei of cells in the MoS 2 NDs group (Fig. 8C).The protein expression of NR4A2 also increased in a dose-and time-dependent manner (Fig. 8D-G).Immunofluorescence staining of mice muscle sections after HLI showed higher NR4A2 + signaling in the CD31 + area in the MoS 2 NDs group (Fig. 8H).All the above data demonstrated that MoS 2 NDs significantly upregulated NR4A2 in vitro and in vivo.To explore whether NR4A2 mediates MoS 2 NDs-induced autophagy, we constructed a small interfering RNA of NR4A2 (si-NR4A2) and confirmed its ability to knockdown the protein expression level of NR4A2 in HUVECs (Figure S38).We then evaluated the impact of knocking down NR4A2 on autophagy.When exposed to HG/Hypo conditions with MoS 2 NDs, there was a significant decrease in autophagy in the si-NR4A2 group, which was demonstrated by a decrease in the LC3 II/I ratio, an increase in the p62 level, and a decrease in the Lamp1 level (Fig. 8I-J).The RFP-GFP-LC3 assay also showed fewer red dots (autophagolysosome) in the si-NR4A2 group, indicating the inhibition of autophagy (Fig. 8K-L, Figure S39).These data confirmed the involvement of NR4A2 in autophagy activation.

Nuclear receptor NR4A2 regulated autophagy by transcriptionally activating autophagy-associated genes ATG4B, ATG9B, and ATG13
Despite substantial evidence from previous studies reporting the involvement of NR4A2 in the regulation of autophagy [72,73], few studies have investigated the definitive mechanism by which NR4A2 regulates autophagy.As a transcription factor, NR4A2 binds to DNA fragments.Therefore, we analyzed the ChIPseq of the NR4A2 from the Gene Expression Omnibus database.The ChIP-seq analysis identified 4799 binding peaks, most of which (47.57%) were located in the promoter regions (Fig. 9A).Genes matched by the binding peaks in the promoter regions were enriched in the autophagy-related pathway (Fig. 9B).Moreover, we were surprised to find that NR4A2 bound to the promoter regions of ATG4B, ATG9B, and ATG13, which played roles in directly regulating autophagosome formation and autophagosome-lysosome fusion (Fig. 9C) [80][81][82].The most significant NR4A2-binding motif in ATG4B, ATG9B, and ATG13 was also identified by ChIP-seq results (Fig. 9D). Figure 9E comprehensively illustrates how MoS 2 NDs upregulate NR4A2 after entering the endothelial cells and how NR4A2 activates autophagy.The western blotting results in Fig. 9F demonstrated that MoS 2 NDs could upregulate the protein expression of ATG4B, ATG9B, and ATG13 along with NR4A2 under HG/Hypo conditions.However, MoS 2 NDs did not upregulate the expression of the three factors when the level of NR4A2 was reduced by small interfering RNA (Fig. 9F-G), indicating that the increase in levels of ATG4B, ATG9B, and ATG13 can be attributed to the upregulation of NR4A2.In summary, MoS 2 NDs increase NR4A2 expression via cAMP/PKA signaling and increase NR4A2 binding to the promoter regions of ATG4B, ATG9B, and ATG13, thereby increasing the expression of these factors and activating autophagy.Therefore, based on the aforementioned results, we have elucidated a molecular mechanism by which MoS 2 nanomaterials promote endothelial cell autophagy.
Collectively, we demonstrated the potential effects of MoS 2 NDs on enhancing collateral formation in T2DM patients.MoS 2 NDs have the advantages of rapid metabolism, low body retention, and high biosafety.Through its antioxidant-like enzyme activity and activation of autophagy, it alleviates endothelial cell dysfunction and thus promotes endothelial angiogenesis in diabetes.
Existing approaches for promoting angiogenesis include pro-angiogenic factor therapy and cell therapy [11,16].Pro-angiogenic factor therapy aims to activate signaling pathways related to angiogenesis, while cell therapy involves the supplementation of cells with angiogenic capabilities to promote angiogenesis.However, these therapies are associated with high costs, limited availability, and susceptibility to degradation [11,12].Additionally, they have inherent deficiencies in diabetes treatment due to impaired angiogenic pathways and the detrimental effects of a high glucose environment on transplanted cells [14,15,17].Conversely, MoS 2 NDs offer a convenient synthesis method, use inexpensive raw materials (Fig. 1), and have demonstrated excellent stability (Figure S3, S6, S14).Moreover, these nanodots specifically treat diabetes-induced oxidative stress (Fig. 3G-H) and the inhibition of autophagy (Fig. 7A-C), thereby restoring endothelial cell function to promote angiogenesis.These advantages highlight the immense potential of MoS 2 NDs compared to existing therapies.
Although previous studies have demonstrated the wide-ranging applications of MoS 2 -based nanomaterials in the field of biomedicine [83][84][85], our research has made progress specifically in the following two aspects: First, we explored the potential of MoS 2 to promote angiogenesis by activating autophagy and uncovered its underlying molecular mechanisms.Firstly, we applied autophagy inhibitors to demonstrate that MoS 2 NDs promote angiogenesis through the activation of autophagy (Fig. 7E-J).Furthermore, RNA sequencing and molecular biology techniques identified NR4A2 as a potential transcription factor mediating autophagy activated by MoS 2 NDs (Fig. 8A-L).Experimental validation revealed that NR4A2 is upregulated by MoS 2 NDs through the activation of the cAMP/PKA signaling pathway (Fig. 8M-N).ChIP-seq analysis of NR4A2 demonstrated its binding to the promoters of ATG4B, ATG9B, and ATG13, promoting their expression and activation of autophagy (Fig. 9).Thus, our study revealed the molecular mechanism of MoS2 NDs activating autophagy through cAMP/ PKA-NR4A2.
Second, we propose a specific therapeutic strategy for impaired neovascularization in diabetes resulting from ROS overload and autophagy Inhibition.Two key factors are closely associated with impaired angiogenesis in diabetes: (1) excessive levels of ROS and (2) impaired autophagy [18,19].By leveraging the dual therapeutic properties of MoS 2 NDs (antioxidant and autophagy activation), we aim to mitigate the detrimental effects of ROS and enhance autophagy, thus promoting angiogenesis with diabetes.This treatment strategy and its demonstrated effectiveness in the article also hold promise for the treatment of a range of complications caused by diabetes, such as diabetic nephropathy, diabetic neuropathy, and diabetic vascular disorders, which are associated with high levels of oxidative stress and autophagic damage.
Nevertheless, this study has some limitations.First, the ultra-small size of MoS 2 NDs facilitates their clearance and metabolism, which improves biosafety but shortens the circulation time of MoS 2 NDs, thereby limiting their effect.Therefore, our future study will employ an engineering strategy to coat biomimetic cell membranes with MoS 2 NDs to prolong their circulation time [86].Second, the size, thickness, and surface modification may affect the activity of the MoS 2 NDs.This study highlights the prospects of applying the MoS 2 NDs for treating T2DM with atherosclerotic diseases and only applies one type of reported MoS 2 NDs to confirm their autophagy activation and ROS scavenging properties.Therefore, further studies should focus on activity regulation by using a broader assessment of the different physiochemical properties of MoS 2 NDs.

Conclusions
In summary, the ultra-small MoS 2 NDs were considered a therapeutic nano-agent for impaired collateral growth in T2DM by combining nanocatalysis therapy with autophagy amplification.Moreover, A specific mechanism (cAMP-NR4A2) has been uncovered to substantiate the effect of MoS 2 NDs in promoting autophagy.The clinical potential and applicability of MoS 2 NDs are evident in two distinct aspects.Firstly, the fabricated MoS 2 NDs exhibited remarkable renal clearance efficiency along with superior biosafety.Should future research affirm their biosecurity in larger animals and potentially in human subjects, MoS 2 NDs could unlock significant translational opportunities.Secondly, the therapeutic efficacy of MoS 2 NDs, as evidenced in our study, extends beyond the realm of diabetes complications.It opens avenues for promising treatments in a spectrum of disorders characterized by deficits in autophagy and elevated oxidative stress.This broadens the scope of MoS 2 NDs, positioning these ultra-small bioactive nanodots as a kind of versatile candidate in the landscape of therapeutic interventions.

Fig. 1
Fig. 1 Schematic showing MoS 2 NDs mediated diabetic neovascularization.The illustration shows that MoS 2 NDs promote angiogenesis by restoring the blood flow of the lower limb in diabetic mice after HLI, as well as facilitating endothelial cell migration, tube formation, and proliferation but inhibiting apoptosis.The antioxidant enzyme activity and autophagy activation properties of MoS 2 NDs have been proposed as potential mechanisms for improving impaired angiogenesis in diabetes

Fig. 2
Fig. 2 Characterization of synthesized MoS 2 NDs (A) TEM image of MoS 2 NDs, inset shows HRTEM image of MoS 2 NDs.The red arrows indicate the boundary around the clear lattice fringe.(B) Size distribution of MoS 2 NDs in PBS.(C) The UV to vis absorption spectra of MoS 2 NDs.(D) XPS data showing the survey spectrum and (E) spectrum of Mo 3d and (F) spectrum of S 2p.(G) Representative images of bio-TEM of MoS 2 NDs within HUVECs under NG conditions and HG/Hypo conditions.(H) The Mo level in HUVECs under NG conditions and HG/Hypo conditions, n = 3 per group.P values were calculated by two-way ANOVA with Tukey's post hoc test.(I) Representative images of intracellular uptake of MoS 2 NDs from different time points using fluorescence microscopy.(J) Intracellular uptake of MoS 2 NDs by flow cytometry and quantification of fluorescence intensity of intracellular MoS 2 NDs, n = 3, and P values were calculated by one-way ANOVA.All data are expressed as mean ± SD.

Fig. 3
Fig. 3 ROS scavenging ability of MoS 2 NDs (A) Schematic illustration of enzyme-mimicking activities of MoS 2 NDs.(B) ESR spectra of DMPO/•OH adducts obtained from samples with Fenton, Fenton and MoS 2 NDs, MoS 2 NDs and DMPO, and single DMPO.(C) ESR spectra of DMPO/•OH adducts obtained from different concentrations of MoS 2 NDs.The ROS scavenging capacity of MoS 2 NDs, including exterminating (D) •OH, (E) •O 2 -, and (F) H 2 O 2 (50 mM) at different concentrations of Mo, n = 3. P values were calculated by one-way ANOVA.(G) Representative images of HUVECs with intracellular ROS probe, DCFH-DA, from different treatments.(H) DCFH-DA fluorescence by flow cytometry and quantification of DCFH-DA fluorescence intensity, n = 5, each group, and P values were calculated by two-way ANOVA with Tukey's post hoc test.All data are expressed as mean ± SD.

Fig. 4
Fig. 4 MoS 2 NDs improved collateral formation in diabetic mice.(A) Illustration of time points of MoS 2 NDs injection and laser Doppler imaging in diabetic mice.(B) Representative H&E images of muscles harvested at the indicated time with PBS and MoS 2 NDs.(C) Quantification of the regenerating area at different time points, n = 5, and P values were calculated by two-way ANOVA with Bonferroni post-hoc test.(D) Quantification of the necrotic area at indicated times from H&E staining, n = 5, and P values were calculated by two-way ANOVA with Bonferroni post-hoc test.(E) Representative images of hindlimb blood perfusion with PBS and MoS 2 NDs at indicated times.(F) Quantification of hindlimb blood perfusion at the indicated times, n = 5, and P values were calculated by two-way ANOVA with Bonferroni post-hoc test.(G) Representative DHE staining images and quantification of DHE + area on day 7, n = 9, P values were calculated by t-test.(H) Representative CD31 + /α-SMA + immunofluorescent images and quantification of CD31 + area and α-SMA area on transverse cross sections on day 7, n = 9, and P values were calculated by t-test.All data are presented as the mean ± SD.

Fig. 5
Fig. 5 MoS 2 NDs alleviated endothelial cell angiogenic dysfunction induced by HG/Hypo.(A) Illustration of transcriptomics of HUVECs treated with MoS 2 NDs and PBS under HG/Hypo conditions.(B) Pathways enriched by differentially expressed genes using GO enrichment analysis.(C) Representative tube formation images of HUVECs with NG + PBS, NG + MoS 2 NDs, HG/Hypo + PBS, and HG/Hypo + MoS 2 NDs.(D) Total vessel length per field, and (E) vessel area per field, n = 4, and P values were calculated by two-way ANOVA with Tukey's post hoc test.(F) Transwell assay of HUVECs and (G) cell migration rate with different treatments, n = 4, and P values were calculated by two-way ANOVA with Tukey's post hoc test.(H) Wound healing assay of HUVECs and (I) cell migration rate with different treatments, n = 4, and P values were calculated by two-way ANOVA with Tukey's post hoc test.All data are expressed as mean ± SD.

Fig. 6
Fig. 6 MoS 2 NDs inhibited cell apoptosis induced by HG/Hypo and promoted endothelial cell proliferation.(A) GSEA plot and heatmap of proliferationassociated genes.(B) GSEA plot and heatmap related to apoptosis.(B) Cell cycle analysis using flow cytometry with different treatments.(D) Percentage of HUVECs on different cell phases, n = 3, and P values were calculated by two-way ANOVA with Tukey's post hoc test.(E) EdU staining on HUVECs and (F) percentage of EdU + cells, n = 4, and P values were calculated by two-way ANOVA with Tukey's post hoc test.(G) Annexin V/PI flow cytometric detection with different treatments.(H) Percentage of apoptotic cells with different treatments, n = 3, and P values were calculated by two-way ANOVA with Tukey's post hoc test.(I) p16 and p21 expressions on HUVECs and (J) quantification of p16 and p21 protein levels with different treatments, n = 3, and P values were calculated by two-way ANOVA with Tukey's post hoc test.(K) The expressions of apoptosis-associated factors: cleaved caspase-3, Bax, Bcl-2, and (L) quantification of these protein expression levels with different treatments, n = 3, and P values were calculated by two-way ANOVA with Tukey's post hoc test.(M) Representative images of JC-1 staining showing early apoptosis of HUVECs with different treatments.All data are expressed as mean ± SD.

Fig. 7
Fig. 7 MoS 2 NDs reversed the inhibition of endothelial autophagy induced by HG/Hypo.(A)GSEA plot and heatmap of genes related to autophagy.(B) Representative images of a single cell expressing RFP-GFP-LC3 following different treatments.(C) The expressions of autophagy-related factors: LC3, p62, Lamp1.(D) Quantification of protein expression levels with different treatments, n = 3, and P values were calculated by two-way ANOVA with Tukey's post hoc test.(E) EdU staining, (F) wound healing analysis, (G) Transwell analysis, (H) tube formation analysis, (I) Annexin-V/PI flow cytometric detection, and (J) JC-1 staining of HUVECs under HG/Hypo conditions treated with MoS 2 NDs with or without the autophagy inhibitor, Baf A1, n = 3, and P values were calculated by t-test.All data are expressed as mean ± SD.

Fig. 8
Fig. 8 MoS 2 NDs on upregulating autophagy-related transcription factors (TFs) NR4A2 via cAMP/PKA signaling.(A) Potential TFs regulating MoS 2 NDsmediated autophagy.(B) Quantification of the mRNA expression levels of TFs with different treatments, n = 3, and P values were calculated by one-way ANOVA with Tukey's post hoc test.(C) Localization of NR4A2 on HUVECs under HG/Hypo conditions with PBS and MoS 2 NDs, measured by fluorescence microscopy.(D) The protein expression levels of NR4A2 with MoS 2 NDs in a dose-dependent manner and (E) time-dependent manner.(F) Quantification of NR4A2 protein expression levels with MoS 2 NDs in a dose-dependent manner and (G) in a time-dependent manner, n = 3, and P values were calculated by one-way ANOVA.(H) Representative images and quantification of the NR4A2 expressions on CD31 + vessels with PBS and MoS 2 ND in vivo.(I) The protein expression levels of LC3, p62, Lamp1, and NR4A2 of HUVECs with si-Ctrl and si-NR4A2 under HG/Hypo conditions treated with MoS 2 NDs.(J) Quantification of protein expression levels, n = 3, and P values were calculated by t-test.(K) Representative images of a single cell expressing RFP-GFP-LC3 with si-Ctrl and si-NR4A2.(L) The quantification of autophagosome (yellow dots) and autophagolysosome (red dots) and the proportion of autophagolysosome in all LC3 puncta, n = 6, and P values were calculated by one-way ANOVA with Tukey's post hoc test and t-test.(M) The related expressions of cAMP, p-PKA, and PKA with PBS and MoS 2 NDs under hypoxia/high glucose condition, n = 3, per group, and P values were calculated by t-test.(N) The related expressions of cAMP, p-PKA, and PKA under hypoxia/high glucose treated with MoS 2 NDs with or without the cAMP inhibitor, SQ22536, n = 3, per group, and P values were calculated by t-test.All data are expressed as mean ± SD.

Fig. 9
Fig. 9 Nuclear receptor NR4A2 activated autophagy by up-regulating autophagy-associated genes ATG4B, ATG9B, and ATG13.(A)The positions of NR4A2binding peaks on a genome-wide scale in ChIP-seq results.(B) The GO enrichment analysis for NR4A2 occupancy.(C) Gene tracks of NR4A2 enrichment by ChIP-seq analysis at core promoter regions of ATG4B, ATG9B, and ATG13.(D) The most significant NR4A2-binding motif of ATG4B, ATG9B, and ATG13 identified by ChIP-seq results.(E) Schematic illustration of MoS 2 NDs-mediated autophagy activation in HUVECs.(F) The related expressions of ATG4B, ATG9B, and ATG13 with PBS and MoS 2 NDs with or without NR4A2 knockdown.(G) Quantification of protein expression levels with different treatments, n = 3, per group, and P values were calculated by one-way ANOVA with Tukey's post hoc test.All data are expressed as mean ± SD. 2